UDP-xylose synthases (UXS) polynucleotides, polypeptides and uses thereof

ABSTRACT

The present disclosure concerns methods and compositions relating to UXS polypeptides and/or nucleic acids encoding UXS polypeptides. In certain claims, the methods and compositions are of use to improve digestibility and/or ease of grain processing. Such improvements relate to a modulation in arabinoxylan and/or hemicellulose content in transgenic plants. Such plants can, for example, comprise one or more nucleic acid sequences that inhibit expression of one or more UDP-Xylose Synthase (USX) genes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/195,929 filed Aug. 2, 2011 and also claims priority to and thebenefit of U.S. patent application Ser. No. 12/486,786 filed Jun. 18,2009, now U.S. Pat. No. 8,017,831 issued on Sep. 13, 2011, U.S. patentapplication Ser. No. 11/614,098 filed Dec. 21, 2006, now U.S. Pat. No.7,592,505 issued Sep. 22, 2009 and U.S. Provisional Patent ApplicationNo. 60/755,253, filed Dec. 30, 2005, all of which are hereinincorporated in their entirety by reference.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for grainimprovement and/or improved digestibility of transgenic plants. Incertain embodiments, these plants can contain a UDP-xylose synthase(UXS) nucleic acid that inhibits arabinoxylan and/or hemicelluloseproduction resulting in improved digestibility and/or ease of grainprocessing. In other embodiments, these plants can contain a UXS nucleicacid over-expressed to increase arabinoxylan and/or hemicelluloseproduction resulting in, for example, harder pericarp when expressed inseed.

BACKGROUND OF THE INVENTION

Primary plant cell walls contain approximately 15-20% hemicellulose inthe vegetative tissue and 40-60% in the grain.

Hemicellulose in monocot walls consists mainly of arabinoxylan (alsoreferred to as glucurono-arabinoxylan or pentosans), a branched polymerconsisting of a beta-gylosyl backbone decorated with arabinosyl andglucuronosyl residues. This polymer is often referred to as arabinoxylanbecause of the relatively low proportion of glucuronosyl residues.Carpita, (1996) Annual Review of Plant Physiol. and Plant MolecularBiology 476:445-476.

Dicot cell walls contain xyloglucan as a major hemicellulosic polymer.Xyloglucan is also a branched polymer consisting of a linearbeta-glycosyl backbone decorated with xylosyl residues, some of whichare substituted with galactosyl residues.

Cell walls of corn grain constitute about 6-8% of the total dry weightof the seed. Whistler, et al., Hemicelluloses In Industrial Gums:Polysaccharides and Their Derivatives, San Diego Academic Press, pp.295-308 (1993). The grain cell wall contains 45-65% arabinoxylan, theremainder being mainly cellulose. Arabinoxylans are considered to beanti-nutritional components of animal feed because they can absorb largeamounts of water. This leads to increased viscosity and possiblesequestering of other digestible feed components, such as starch andpolypeptides, away from digestive enzymes.

Arabinoxylans are known to lower the food conversion ratio (FCR) ofanimal feed. Studies where fungal xylanase were included incereal-derived feed showed improved FCR and weight gain in broilers andpigs. Veldman and Vahl, (1994) British Poultry Science 35:537-550. Theobserved improvement in FCR and weight gain was higher than expectedfrom arabinoxylan breakdown and digestion alone. Thus, arabinoxylanappears to reduce the digestion of other feed components. An unresolvedneed exists for methods and compositions to reduce the concentration ofarabinoxylan (and thus hemicellulose) in grain.

Cellulose microfibrils have the highest tensile strength of any of theother cell wall polymers. Increasing the cellulose/arabinoxylan ratio inthe cell wall should lead to a harder pericarp and improved ability tohandle the grain.

About 12% of the corn processed through wet-milling is fiber (see,Johnson and Can, (2003) Wet milling: the basis of corn biorefineries; inCorn: Chemistry and Technology. P J White and L A Johnson (eds). AACC,St. Paul, Minn. pp. 449-494). Of this 12%, approximately 4% is coarsefiber (mainly pericarp-derived) and the rest is fine fiber. While coarsefiber consists of approximately 9% starch, fine fiber can have up to 30%starch, constituting 2.5% of the total dry mass. The other majorcomponent of fine fiber is arabinoxylan, which makes up to 21% of thisfraction. Some starch can become intricately associated witharabinoxylan, making it difficult to extract during wet-milling. A needexists for a method to reduce the concentration of arabinoxylan (andthus hemicellulose) in grain to improve starch extractability.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Terms that are not otherwise defined herein are used in accordance withtheir plain and ordinary meaning.

Units, prefixes and symbols can be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges recitedwithin the specification are inclusive of the numbers defining the rangeand include each integer within the defined range. Amino acids can bereferred to herein by either their commonly known three letter symbolsor by the one-letter symbols recommended by the IUPAC-IUB BiochemicalNomenclature Commission. Nucleotides, likewise, can be referred to bytheir commonly accepted single-letter codes. Unless otherwise providedfor, software, electrical and electronics terms as used herein are asdefined in The New IEEE Standard Dictionary of Electrical andElectronics Terms (5th edition, 1993). The terms defined below are morefully defined by reference to the specification as a whole.

As used herein, “a” or “an” can mean one or more than one of an item.

“UXS polynucleotide” refers to a nucleic acid construct that comprisesat least part or all of the UXS nucleic acid sequence, non-limitingexamples of which are provided in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 and15. A “polynucleotide” can be single, double and/or triple stranded andcan comprise part or all of a native or modified UXS nucleic acidsequence. A “polynucleotide” can be designed to produce an inhibitoryRNA that decreases expression of the endogenous UXS sequences. A“polynucleotide” can be of any size, from a short oligonucleotide to afull length UXS gene. A UXS “polynucleotide” sequence can be in thesense or anti-sense orientation. A “polynucleotide” can be of genomic orcDNA sequence.

The present invention also provides isolated polynucleotide sequencescomprising transcriptional units for gene over-expression andgene-suppression that have been used either as single units or incombination as multiple units to transform plant cells.

A “transgenic plant” is one that contains an introduced segment ofnucleic acid, such as a UXS nucleic acid. The introduced nucleic acidcan encode a native UXS polynucleotide sequence or a modifiedpolynucleotide sequence of any length, up to a full-length sequence. Anymethod known in the art can be used to transform one or more plant cellsand regenerate a transgenic plant.

As used herein “operably linked” refers to a functional linkage betweena promoter and/or other regulatory element and a second nucleic acidsequence, wherein the promoter initiates and mediates transcription ofthe second sequence.

The term “isolated” refers to material, such as a nucleic acid or aprotein, which is: (1) substantially or essentially free from componentswhich normally accompany or interact with the material as found in itsnaturally occurring environment or (2) if the material is in its naturalenvironment, the material has been altered by deliberate humanintervention to a composition and/or placed at a locus in the cell otherthan the locus native to the material.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cellsand progeny of same. “Plant cell”, as used herein includes, withoutlimitation, seeds, suspension cultures, embryos, meristematic regions,callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen,and microspores. The class of plants which can be used in the claimedmethods is generally as broad as the class of higher plants amenable totransformation techniques, including both monocotyledonous anddicotyledonous plants.

By “fragment” is intended a portion of the nucleotide sequence or aportion of the amino acid sequence and hence protein encoded thereby.Fragments of a nucleotide sequence can encode protein fragments thatretain the biological activity of the native nucleic acid, i.e.,“functional fragments”.

Alternatively, fragments of a nucleotide sequence that can be useful ashybridization probes may not encode fragment proteins retainingbiological activity. Thus, fragments of a nucleotide sequence aregenerally greater than 25, 50, 100, 150, 200, 250, 300, 350, 400, 450,500, 600 or 700 nucleotides and up to and including the entirenucleotide sequence encoding the proteins of the invention. Generallythe probes are less than 1000 nucleotides and often less than 500nucleotides.

Similarly, fragments of a nucleotide sequence that are useful forgenerating cells, tissues or plants transiently or permanentlysuppressing a gene or genes may not encode fragment proteins retainingbiological activity. Fragments may be in sense or antisense orientation,reverse orientation, complementary or a combination thereof. Thus, forexample, fragments of such nucleotide sequence may range from at leastabout 10 nucleotides, to greater than 25, 50, 100, 200, 300, 400, 500,600 or 700 nucleotides and up to and including the full-lengthnucleotide sequence-encoding native UXS proteins.

By “variants” is intended substantially similar sequences. Generally,nucleic acid sequence variants of the invention will have at least 60%,65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to thenative nucleotide sequence, wherein the % sequence identity is based onthe entire sequence and is determined by GAP 10 analysis using defaultparameters. Generally, polypeptide sequence variants of the inventionwill have at least about 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% sequence identity to the native protein,wherein the % sequence identity is based on the entire sequence and isdetermined by GAP 10 analysis using default parameters. GAP uses thealgorithm of Needleman and Wunsch (J. Mol. Biol. 48:443-453, 1970) tofind the alignment of two complete sequences that maximizes the numberof matches and minimizes the number of gaps.

As used herein “transformation” can include stable transformation andtransient transformation. Unless otherwise stated, “transformation”refers to stable transformation.

As used herein “stable transformation” refers to the transfer of anucleic acid fragment into a genome of a host organism (this includesboth nuclear and organelle genomes) resulting in genetically stableinheritance. In addition to traditional methods, stable transformationincludes the alteration of gene expression by any means includingchimeraplasty or transposon insertion.

As used herein “transient transformation” refers to the transfer of anucleic acid fragment or protein into the nucleus (or DNA-containingorganelle) of a host organism resulting in gene expression withoutintegration and stable inheritance.

As used herein, the term “co-suppression” is used to collectivelydesignate gene silencing methods based on mechanisms involving theexpression of antisense, sense RNA molecules, aberrant RNA molecules,double-stranded RNA molecules, micro RNA molecules and the like.

“Non-ruminant animal” means an animal with a simple stomach divided intothe esophageal, cardia, fundus and pylorus regions. A non-ruminantanimal additionally implies a species of animal without a functionalrumen. A rumen is a section of the digestive system where feedstuff/foodis soaked and subjected to digestion by microorganisms before passing onthrough the digestive tract. This phenomenon does not occur in anon-ruminant animal. The term non-ruminant animal includes, but is notlimited to, humans, swine, poultry, cats and dogs.

UDP-Xylose Synthases (UXS) in Plants

UDP-xylose (Xyl) is an essential sugar donor for the synthesis ofhemicellulose, glycoproteins and oligosaccharides. UDP-Xyl also feedbackinhibits upstream enzymes. In plants, the major route by which UDP-Xylis produced is via UDP-Glucose. The biosynthesis of UDP-Xyl is catalyzedby different UDP-xylose synthase (UXS) isozymes, all of which convertUDP-GlcA to UDP-Xyl by NADH dependent decarboxylation of UDP-glucuronicacid.

A plant UDP-xylose synthase (UXS) was purified from pea by peptidesequencing and enzyme activity was demonstrated with the recombinantpolypeptide. The A. thaliana and rice genomes each contain six putativeUXS genes that are highly similar to both the pea and fungal UXS genes.The sub-cellular location of UXS activities varies in different species.For example, the only UXS enzyme from rat is localized to the Golgi,while the sole UXS of Cryptococcus is cytosolic. In plants, activitiesof UXS enzymes are either soluble or membrane associated. Three of theArabidopsis UXS isoforms (AtUXS 3, 5, 6) appear to be cytosolic and theother three (AtUxs 1, 2, 4) are likely to reside in the endomembranesystem. In addition to six UXS isoforms, two A. thaliana genes that aredistantly related to the UXS gene family apparently encodeUDP-D-apiose/UDP-D-xylose synthase (AXS). Purified recombinant AXS1generates UDP-D-xylose and UDP-D-apiose using UDP-D-glucuronic acid asits exclusive substrate.

Nucleic Acids

In various embodiments, nucleic acids of the invention can encode a UXSpolypeptide sequence, an inhibitory RNA or another type of UXSmacromolecule. The nucleic acid can be derived from genomic DNA,complementary DNA (cDNA), RNA or synthetic DNA. Where incorporation intoan expression cassette is desired, the nucleic acid can also compriseone or more introns.

A nucleic acid includes single-stranded and double-stranded ortriple-stranded molecules, as well as DNA, RNA, chemically modifiednucleic acids and nucleic acid analogs. It is contemplated that anucleic acid can be of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, about 110, about 120,about 130, about 140, about 150, about 160, about 170, about 180, about190, about 200, about 210, about 220, about 230, about 240, about 250,about 275, about 300, about 325, about 350, about 375, about 400, about425, about 450, about 475, about 500, about 525, about 550, about 575,about 600, about 625, about 650, about 675, about 700, about 725, about750, about 775, about 800, about 825, about 850, about 875, about 900,about 925, about 950, about 975, about 1000, about 1100, about 1200,about 1300, about 1400, about 1500, about 1750, about 2000, about 2250,about 2500 or greater nucleotide residues in length, up to a full lengthUXS gene.

UXS polypeptides can be encoded by any nucleic acid sequence thatencodes the appropriate amino acid sequence. The design and productionof nucleic acids encoding a desired amino acid sequence is well known tothose of skill in the art, using standardized codon tables (see, Table 1below). The codons selected for encoding each amino acid can be modifiedto optimize expression of the nucleic acid in the host cell of interest,for example by using codons optimized for expression in maize. Codonpreferences for various species of host cell are well known in the art.

TABLE 1 Amino Acid Codons Alanine Ala GCA  GCC GCG  GCU Cysteine Cys UGCUGU Aspartic acid Asp GAc GAU Glutamic acid Glu GAA GAG PhenylalaninePhe UUC UUU Glycine Gly GGA GGC GGG GGU Histidine His CAC CAU IsoleucineIle AUA AUC AUU Lysine Lys AAA AAG Leucine Leu UUA UUG CUA CUC CUG CUUMethionine Met AUG Asparagine Asn AAC AAU Proline Pro CCA CCC CCG CCUGlutamine Gln CAA CAG Arginine Arg AGA AGG CGA CGC CGG CGU Serine SerAGC AGU UCA UCC UCG UCU Threonine Thr ACA ACC ACG ACU Valine Val GUA GUCGUG GUU Tryptophan Trp UGG Tyrosine Ty UAC UAUNucleic Acids

Isolated nucleic acids can be made by any method known in the art, forexample using standard recombinant methods, synthetic techniques, orcombinations thereof. In some embodiments, the nucleic acids can becloned, amplified, or otherwise constructed.

The nucleic acids can conveniently comprise sequences in addition to UXSnucleic acid sequences. For example, a multi-cloning site comprising oneor more endonuclease restriction sites can be added. Regulatorysequences can be added to promote expression of the nucleic acid.Translatable sequences can be inserted to aid in the isolation ofexpressed polypeptides. For example, a hexa-histidine marker sequenceprovides a convenient means to purify tagged polypeptides. A nucleicacid can be attached to a cassette, adapter or linker for cloning and/orexpression of a nucleic acid. Additional sequences can be added to suchcloning and/or expression sequences to optimize their function incloning and/or expression, to aid in isolation of the nucleic acid, orto improve the introduction of the nucleic acid into a cell. Use ofcloning cassettes, expression cassettes, adapters and linkers is wellknown in the art.

Isolated nucleic acids, such as RNA, cDNA, genomic DNA, or a hybridthereof, can be obtained from plant biological sources using any numberof cloning methodologies known in the art. In some embodiments,oligonucleotide probes which selectively hybridize, under stringentconditions, to the nucleic acids are used to identify a sequence in acDNA or genomic DNA library. Methods for isolation of mRNA andconstruction of cDNA and genomic libraries are known and any such knownmethods can be used. (See, e.g., Plant Molecular Biology: A LaboratoryManual, Clark, Ed., Springer-Verlag, Berlin (1997); Current Protocols inMolecular Biology, Ausubel, et al., Eds., Greene Publishing andWiley-Interscience, New York (1995); Carninci, et al., (1996) Genomics37:327-336; Ko, (1990) Nucl. Acids. Res. 18(19):5705-5711; Sambrook, etal., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Vols. 1-3 (1989); Methods in Enzymology, Vol. 152, Guide toMolecular Cloning Techniques, Berger and Kimmel, Eds., San DiegoAcademic Press, Inc. (1987).]

cDNA or genomic libraries, transgenic cell lines, transgenic plants ortissues or native plants or tissues can be screened for the presenceand/or expression levels of UXS nucleic acids using a probe based uponone or more UXS sequences, such as those disclosed in the presentinvention. Probes also can be used to hybridize with genomic DNA or cDNAsequences to isolate homologous genes in the same or different plantspecies. Various degrees of stringency of hybridization can be employedin the assay. As the conditions for hybridization become more stringent,there must be a greater degree of complementarity between the probe andthe target for duplex formation to occur. The degree of stringency canbe controlled by temperature, ionic strength, pH and/or the presence ofa partially denaturing solvent such as formamide. For example, thestringency of hybridization is conveniently varied by changing thepolarity of the reactant solution through manipulation of theconcentration of formamide within the range of 0% to 50%. The degree ofcomplementarity (sequence identity) required for detectable binding willvary in accordance with the stringency of the hybridization mediumand/or wash medium. The degree of complementarity will optimally be 100percent; however, minor sequence variations in the probes and primerscan be compensated for by reducing the stringency of the hybridizationand/or wash medium.

Typically, stringent hybridization conditions will be those in which thesalt concentration is less than about 1.5 M Na ion, typically about 0.01to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions can also be achieved with theaddition of destabilizing agents such as formamide.

Exemplary low stringency conditions include hybridization with a buffersolution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecylsulfate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 Mtrisodium citrate) at 50° C. Exemplary moderate stringency conditionsinclude hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37°C., and a wash in 0.5× to 1×SSC at 55° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C. and a wash in 0.1×SSC at 60° C. Typically the time ofhybridization is from 4 to 16 hours.

An extensive guide to the hybridization of nucleic acids is found inTijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y. (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995). Often, cDNA libraries will benormalized to increase the representation of relatively rare cDNAs.

Nucleic acids of interest can also be amplified using a variety of knownamplification techniques. For instance, polymerase chain reaction (PCR)technology can be used to amplify target sequences directly from genomicDNA or cDNA sequences. PCR and other in vitro amplification methods canalso be useful, for example, to clone nucleic acid sequences that codefor polypeptides to be expressed, to make nucleic acids to use as probesfor detecting the presence of the desired mRNA in samples, for nucleicacid sequencing, or for other purposes. Examples of techniques of usefor nucleic acid amplification are found in Berger, Sambrook, andAusubel, as well as Mullis, et al., U.S. Pat. No. 4,683,202 (1987) and,PCR Protocols A Guide to Methods and Applications, Innis, et al., Eds.,Academic Press Inc., San Diego, Calif. (1990). The T4 gene 32polypeptide (Boehringer Mannheim) can be used to improve yield of longPCR products. PCR-based screening methods have been disclosed. (See,e.g., Wilfinger, et al., (1997) BioTechniques 22(3):481-486).

Isolated nucleic acids can be prepared by direct chemical synthesis bymethods such as the phosphotriester method of Narang, et al., (1979)Meth. Enzymol. 68:90-99; the phosphodiester method of Brown, et al.,(1979) Meth. Enzymol. 68:109-151; the diethylphosphoramidite method ofBeaucage, et al., (1981) Tetra. Lett. 22:859-1862; the solid phasephosphoramidite triester method of Beaucage and Caruthers, (1981) Tetra.Letts. 22(20):1859-1862, using an automated synthesizer as inNeedham-VanDevanter, et al., (1984) Nucleic Acids Res. 12:6159-6168 orby the solid support method of U.S. Pat. No. 4,458,066. Chemicalsynthesis generally produces a single stranded oligonucleotide. This canbe converted into double stranded DNA by hybridization with acomplementary sequence or by polymerization with a DNA polymerase usingthe single strand as a template. While chemical synthesis of DNA is bestemployed for sequences of about 100 bases or less, longer sequences canbe obtained by the ligation of shorter sequences.

A variety of cross-linking agents, alkylating agents and radicalgenerating species can be used to bind, label, detect and/or cleavenucleic acids. For example, Vlassov, et al., (1986) Nucleic Acids Res14:4065-4076, disclose covalent bonding of a single-stranded DNAfragment with alkylating derivatives of nucleotides complementary totarget sequences. A report of similar work by the same group is that byKnorre, et al., (1985) Biochimie 67:785-789. Iverson and Dervan alsoshowed sequence-specific cleavage of single-stranded DNA mediated byincorporation of a modified nucleotide which was capable of activatingcleavage (J Am Chem Soc (1987) 109:1241-1243). Meyer, et al., (1989) JAm Chem Soc 111:8517-8519 disclose covalent crosslinking to a targetnucleotide using an alkylating agent complementary to thesingle-stranded target nucleotide sequence. A photoactivatedcrosslinking to single-stranded oligonucleotides mediated by psoralenwas disclosed by Lee, et al., (1988) Biochemistry 27:3197-3203. Use ofcrosslinking in triple-helix forming probes was also disclosed by Home,et al., (1990) J Am Chem Soc 112:2435-2437. Use of N4, N4-ethanocytosineas an alkylating agent to crosslink to single-stranded oligonucleotideshas also been disclosed by Webb and Matteucci, (1986) J Am Chem Soc108:2764-2765; Nucleic Acids Res (1986) 14:7661-7674; Feteritz, et al.,(1991) J. Am. Chem. Soc. 113:4000. Various compounds to bind, detect,label and/or cleave nucleic acids are known in the art. See, forexample, U.S. Pat. Nos. 5,543,507; 5,672,593; 5,484,908; 5,256,648 and5,681,941.

Expression Cassettes

Various embodiments concern cassettes comprising UXS nucleic acids,which cassettes can be transformed into a target host cell. Anexpression cassette will typically comprise a nucleic acid operablylinked to transcriptional regulatory elements which will direct thetranscription of the nucleic acid. For example, plant expressioncassettes can include a cloned UXS nucleic acid under thetranscriptional control of 5′ and 3′ regulatory sequences. Expressioncassettes can contain a promoter sequence (e.g., one conferringinducible or constitutive, environmentally- or developmentally-regulatedor cell- or tissue-specific/selective expression), a transcriptioninitiation start site, a ribosome binding site, an RNA processingsignal, a transcription termination site and/or a polyadenylationsignal.

The nucleotide sequences for use in the methods of the present inventionare provided in expression cassettes for transcription in the plant ofinterest. Such expression cassettes are provided with a plurality ofrestriction sites for insertion of any sequence of the present inventionto be placed under the transcriptional regulation of the regulatoryregions. The expression cassettes may additionally contain selectablemarker genes.

The expression cassette can include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region,any seed protein sequence of the invention and optionally, atranscriptional and translational termination region functional inplants. The transcriptional initiation region may be native or analogousor foreign or heterologous to the plant host. Additionally, the promotermay be the natural sequence or alternatively a synthetic sequence. By“foreign” is intended that the transcriptional initiation region is notfound in the native plant into which the transcriptional initiationregion is introduced. As used herein, a gene comprises a coding sequenceoperably linked to a transcription initiation region that isheterologous to the coding sequence. Alternatively, a gene comprisesfragments of at least two independent transcripts that are linked in asingle transcription unit.

While it may be preferable to express the sequences using heterologouspromoters, the native promoter sequences may be used. Such constructswould alter expression levels of the proteins in the plant or plantcell. Thus, the phenotype of the plant or plant cell is altered.Alternatively, the promoter sequence may be used to alter expression.For example, the promoter (or fragments thereof) of a UXS sequence ofthe present invention can modulate expression of the native UXS proteinor other closely related proteins.

Use of a termination region is not necessary for proper transcription ofplant genes but may be used as part of an expression construct. Thetermination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,or may be derived from another source. Convenient termination regionsare available from the Ti-plasmid of A. tumefaciens, such as theoctopine synthase and nopaline synthase termination regions. See also,Guerineau, et al., (1991) Mol. Gen. Genet. 262:141-144; Proudfoot,(1991) Cell 64:671-674; Sanfacon, et al., (1991) Genes Dev. 5:141-149;Mogen, et al., (1990) Plant Cell 2:1261-1272; Munroe, et al., (1990)Gene 91:151-158; Ballas, et al., (1989) Nucleic Acids Res. 17:7891-7903and Joshi, et al., (1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, the genes can be synthesized using plant-preferredcodons for improved expression. See, for example, Campbell and Gowri,(1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codonusage. Methods are available in the art for synthesizing plant-preferredgenes. See, for example, U.S. Pat. Nos. 5,380,831 and 5,436,391 andMurray, et al., (1989) Nucleic Acids Res. 17:477-498, hereinincorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats and other such well-characterized sequences thatmay be deleterious to gene expression. The G-C content of the sequencemay be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein, et al., (1989)Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, forexample, TEV leader (Tobacco Etch Virus) (Gallie, et al., (1995) Gene165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology154:9-20), and human immunoglobulin heavy-chain binding protein (BiP)(Macejak, et al., (1991) Nature 353:90-94); untranslated leader from thecoat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al.,(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie,et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York),pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel, etal., (1991) Virology 81:382-385). See also, Della-Cioppa, et al., (1987)Plant Physiol. 84:965-968. Other methods known to enhance translationcan also be utilized, for example, introns, and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues. Marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds,such as glufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). See generally, Yarranton, (1992)Curr. Opin. Biotech. 3:506-511; Christopherson, et al., (1992) Proc.Natl. Acad. Sci. USA 89:6314-6318; Yao, et al., (1992) Cell 71:63-72;Reznikoff, (1992) Mol. Microbiol. 6:2419-2422; Barkley, et al., (1980)in The Operon, pp. 177-220; Hu, et al., (1987) Cell 48:555-566; Brown,et al., (1987) Cell 49:603-612; Figge, et al., (1988) Cell 52:713-722;Deuschle, et al., (1989) Proc. Natl. Acad. Aci. USA 86:5400-5404;Fuerst, et al., (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553;Deuschle, et al., (1990) Science 248:480-483; Gossen, (1993) Ph.D.Thesis, University of Heidelberg; Reines, et al., (1993) Proc. Natl.Acad. Sci. USA 90:1917-1921; Labow, et al., (1990) Mol. Cell. Biol.10:3343-3356; Zambretti, et al., (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Baim, et al., (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman, (1989) Topics Mol. Struc. Biol. 10:143-162;Degenkolb, et al., (1991) Antimicrob. Agents Chemother. 35:1591-1595;Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; Bonin, (1993)Ph.D. Thesis, University of Heidelberg; Gossen, et al., (1992) Proc.Natl. Acad. Sci. USA 89:5547-5551; Oliva, et al., (1992) Antimicrob.Agents Chemother. 36:913-919; Hlavka, et al., (1985) Handbook ofExperimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill, etal., (1988) Nature 334:721-724. Such disclosures are herein incorporatedby reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the present invention.

The use of the term “nucleotide constructs” herein is not intended tolimit the present invention to nucleotide constructs comprising DNA.Those of ordinary skill in the art will recognize that nucleotideconstructs, particularly polynucleotides and oligonucleotides, comprisedof ribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides may also be employed in the methods disclosedherein. Thus, the nucleotide constructs of the present inventionencompass all nucleotide constructs that can be employed in the methodsof the present invention for transforming plants including, but notlimited to, those comprised of deoxyribonucleotides, ribonucleotides,and combinations thereof. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thenucleotide constructs of the invention also encompass all forms ofnucleotide constructs including, but not limited to, single-strandedforms, double-stranded forms, hairpins, stem-and-loop structures and thelike.

Furthermore, it is recognized that the methods of the invention mayemploy a nucleotide construct that is capable of directing, in atransformed plant, the expression of at least one protein, or at leastone RNA, such as, for example, an antisense RNA that is complementary toat least a portion of an mRNA. Alternatively, it is also recognized thatthe methods of the invention may employ a nucleotide construct that isnot capable of directing, in a transformed plant, the expression of aprotein or an RNA.

In addition, it is recognized that methods of the present invention donot depend on the incorporation of the entire nucleotide construct intothe genome, only that the plant or cell thereof is altered as a resultof the introduction of the nucleotide construct into a cell. In oneembodiment of the invention, the genome may be altered following theintroduction of the nucleotide construct into a cell. For example, thenucleotide construct, or any part thereof, may incorporate into thegenome of the plant. Alterations to the genome of the present inventioninclude, but are not limited to, additions, deletions and substitutionsof nucleotides in the genome. While the methods of the present inventiondo not depend on additions, deletions or substitutions of any particularnumber of nucleotides, it is recognized that such additions, deletionsor substitutions comprise at least one nucleotide.

The nucleotide constructs of the invention also encompass nucleotideconstructs that may be employed in methods for altering or mutating agenomic nucleotide sequence in an organism, including, but not limitedto, chimeric vectors, chimeric mutational vectors, chimeric repairvectors, mixed-duplex oligonucleotides, self-complementary chimericoligonucleotides and recombinogenic oligonucleobases. Such nucleotideconstructs and methods of use, such as, for example, chimeraplasty, areknown in the art. Chimeraplasty involves the use of such nucleotideconstructs to introduce site-specific changes into the sequence ofgenomic DNA within an organism. See U.S. Pat. Nos. 5,565,350; 5,731,181;5,756,325; 5,760,012; 5,795,972 and 5,871,984, all of which are hereinincorporated by reference. See also, WO 98/49350, WO 99/07865, WO99/25821 and Beetham, et al., (1999) Proc. Natl. Acad. Sci. USA96:8774-8778, herein incorporated by reference.

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, tissue-preferred or otherpromoters for expression in plants, more preferably a promoterfunctional during seed development.

Such constitutive promoters include, for example, the core promoter ofthe Rsyn7 promoter and other constitutive promoters disclosed in WO99/43838; the core CaMV 35S promoter (Odell, et al., (1985) Nature313:810-812); rice actin (McElroy, et al., (1990) Plant Cell 2:163-171);ubiquitin (Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 andChristensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, etal., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten, et al., (1984)EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and thelike. Other constitutive promoters include, for example, U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463 and 5,608,142.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression or a chemical-repressible promoter, where application ofthe chemical represses gene expression. Chemical-inducible promoters areknown in the art and include, but are not limited to, the maize In2-2promoter, which is activated by benzenesulfonamide herbicide safeners,the maize GST promoter, which is activated by hydrophobic electrophiliccompounds that are used as pre-emergent herbicides, and the tobaccoPR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena, et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis, et al., (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz, et al., (1991) Mol. Gen. Genet. 227:229-237 and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced proteinexpression within a particular plant tissue. Tissue-preferred promotersinclude, but are not limited to: Yamamoto, et al., (1997) Plant J.12(2)255-265; Kawamata, et al., (1997) Plant Cell Physiol.38(7):792-803; Hansen, et al., (1997) Mol. Gen. Genet. 254(3):337-343;Russell, et al., (1997) Transgenic Res. 6(2):157-168; Rinehart, et al.,(1996) Plant Physiol. 112(3):1331-1341; Van Camp, et al., (1996) PlantPhysiol. 112(2):525-535; Canevascini, et al., (1996) Plant Physiol.112(2):513-524; Yamamoto, et al., (1994) Plant Cell Physiol.35(5):773-778; Lam, (1994) Results Probl. Cell Differ. 20:181-196;Orozco, et al., (1993) Plant Mol. Biol. 23(6):1129-1138; Matsuoka, etal., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 andGuevara-Garcia, et al., (1993) Plant J. 4(3):495-505. Such promoters canbe modified, if necessary, for weak expression.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See, Thompson, et al., (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kD zein) and milps (myo-inositol-1-phosphatesynthase; see, U.S. Pat. No. 6,225,529, herein incorporated byreference). The 27 kD gamma-zein is a preferred endosperm-specificpromoter. Glb-1 is a preferred embryo-specific promoter.

For dicots, seed-specific promoters include, but are not limited to,bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, andthe like.

For monocots, seed-specific promoters include, but are not limited to,maize 15 kD zein, 22 kD zein, 27 kD zein, 10 kD delta-zein, waxy,shrunken 1, shrunken 2, globulin 1, etc.

Regulation of Gene Expression

While not critical to the invention, the methods of the inventioncomprise either increasing or decreasing the level of a target geneproduct. Methods for inhibiting gene expression are well known in theart. Although any method know in the art for reducing the level ofprotein in a plant could be used, possible methods for reducing proteininclude, but are not limited to, homology-dependent gene silencing,antisense technology, co-suppression including, for example, RNAinterference (RNAi), micro RNA and the like, site-specificrecombination, site-specific integration, mutagenesis includingtransposon tagging, and biosynthetic competition, homologousrecombination and gene targeting, alone or in combination. Dependingupon the intended goal, the level of at least one seed protein may beincreased, decreased or eliminated.

Catalytic RNA molecules or ribozymes may also be used to inhibitexpression of plant genes. It is possible to design ribozymes thatspecifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA-cleaving activity upon them,thereby increasing the activity of the constructs. The design and use oftarget RNA-specific ribozymes is disclosed, for example, in Haseloff, etal., (1988) Nature 334:585-591.

A “co-suppression” cassette may include 5′ (but not necessarily 3′)regulatory sequences, operably linked to at least one of the sequencesof the invention. Co-supression cassettes used in the methods of theinvention can comprise sequences of the invention in so-called “invertedrepeat” structures. The cassette may additionally contain a second copyof the fragment in opposite direction to form an inverted repeatstructure: Opposing arms of the structure may or may not be interruptedby any nucleotide sequence related or unrelated to the nucleotidesequences of the invention. (See, Fiers, et al., U.S. Pat. No.6,506,559). The transcriptional units are linked to be co-transformedinto the organism. Alternatively, additional transcriptional units canbe provided on multiple over-expression and co-suppression cassettes.

The methods of transgenic co-suppression can be used to reduce oreliminate the level of at least one seed protein in grain. One method oftransgenic co-suppression comprise transforming a plant cell with atleast one transcriptional unit containing an expression cassettecomprising a promoter that drives transcription in the plant operablylinked to at least one nucleotide sequence transcript in the senseorientation encoding at least a portion of the seed protein of interest.Methods for suppressing gene expression in plants using nucleotidesequences in the sense orientation are known in the art. The methodsgenerally involve transforming plants with a DNA construct comprising apromoter that drives transcription in a plant operably linked to atleast a portion of a nucleotide sequence that corresponds to thetranscript of the endogenous gene. Typically, such a nucleotide sequencehas substantial sequence identity to the sequence of the transcript ofthe endogenous gene, at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity overthe entire length of the sequence. Furthermore, portions, rather thanthe entire nucleotide sequence, of the polynucleotides may be used todisrupt the expression of the target gene product. Generally, sequencesof at least 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200 nucleotides orgreater may be used. See, U.S. Pat. Nos. 5,283,184 and 5,034,323, hereinincorporated by reference.

The endogenous gene targeted for co-suppression may be a gene encodingany seed protein that accumulates as a seed protein in the plant speciesof interest, including, but not limited to, the seed genes noted above.For example, where the endogenous gene targeted for co-suppression is aUXS gene disclosed herein, co-suppression is achieved using anexpression cassette comprising a UXS gene sequence or variant orfragment thereof.

Additional methods of co-suppression are known in the art and can besimilarly applied to the instant invention. These methods involve thesilencing of a targeted gene by spliced hairpin RNA's and similarmethods also called RNA interference and promoter silencing (see, Smith,et al., (2000) Nature 407:319-320, Waterhouse and Helliwell, (2003) Nat.Rev. Genet. 4:29-38; Waterhouse, et al., (1998) Proc. Natl. Acad. Sci.USA 95:13959-13964; Chuang and Meyerowitz, (2000) Proc. Natl. Acad. Sci.USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant Phystiol.129:1723-1731 and Patent Applications WO 99/53050; WO 99/49029; WO99/61631; WO 00/49035 and U.S. Pat. No. 6,506,559, each of which isherein incorporated by reference). For the purpose of this invention theterm “co-suppression” is used to collectively designate gene silencingmethods based on mechanisms involving the expression of sense RNAmolecules, aberrant RNA molecules, double-stranded RNA molecules, microRNA molecules and the like.

The expression cassette for co-suppression may also be designed suchthat the sense sequence and the antisense sequence do not correspond toan endogenous RNA In this embodiment, the sense and antisense sequenceflank a loop sequence that comprises a nucleotide sequence correspondingto all or part of the endogenous messenger RNA of the target gene. Thus,it is the loop region that determines the specificity of the RNAinterference. See, for example, International Publication Number WO02/00904, herein incorporated by reference.

In other embodiments of the invention, inhibition of the expression of aprotein of interest may be obtained by RNA interference by expression ofa gene encoding a micro RNA (miRNA). miRNAs are regulatory agentsconsisting of about 22 ribonucleotides. miRNA are highly efficient atinhibiting the expression of endogenous genes. See, for example, Javier,et al., (2003) Nature 425: 257-263, herein incorporated by reference.

For miRNA interference, the expression cassette is designed to expressan RNA molecule that is modeled on an endogenous miRNA gene. The miRNAgene encodes an RNA that forms a hairpin structure containing a22-nucleotide sequence that is complementary to another endogenous gene(target sequence). miRNA molecules are highly efficient at inhibitingthe expression of endogenous genes and the RNA interference they induceis inherited by subsequent generations of plants

In one embodiment, the polynucleotide to be introduced into the plantcomprises an inhibitory sequence that encodes a zinc finger protein thatbinds to a gene encoding a protein of the invention resulting in reducedexpression of the gene. In particular embodiments, the zinc fingerprotein binds to a regulatory region of a UXS gene. In otherembodiments, the zinc finger protein binds to a messenger RNA encoding aseed protein and prevents its translation. Methods of selecting sitesfor targeting by zinc finger proteins have been described, for example,in U.S. Pat. No. 6,453,242 and methods for using zinc finger proteins toinhibit the expression of genes in plants are described, for example, inUS Patent Application Publication Number 2003/0037355; each of which isherein incorporated by reference.

Methods for antisense suppression can be used to reduce or eliminate thelevel of at least one protein of the invention. The methods of antisensesuppression comprise transforming a plant cell with at least oneexpression cassette comprising a promoter that drives expression in theplant cell operably linked to at least one nucleotide sequence that isantisense to a nucleotide sequence transcript of the target protein. By“antisense suppression” is intended the use of nucleotide sequences thatare antisense to nucleotide sequence transcripts of endogenous plantgenes to suppress the expression of those genes in the plant.

Methods for suppressing gene expression in plants using nucleotidesequences in the antisense orientation are known in the art. The methodsgenerally involve transforming plants with a DNA construct comprising apromoter that drives expression in a plant operably linked to at least aportion of a nucleotide sequence that is antisense to the transcript ofthe endogenous gene. Antisense nucleotides are constructed to hybridizewith the corresponding mRNA. Modifications of the antisense sequencesmay be made as long as the sequences hybridize to and interfere withexpression of the corresponding mRNA. In this manner, antisenseconstructions having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to thecorresponding antisense sequences may be used. Furthermore, portions,rather than the entire nucleotide sequence, of the antisense nucleotidesmay be used to disrupt the expression of the target gene. Generally,sequences of at least 10 nucleotides, 50 nucleotides, 100 nucleotides,200 nucleotides or greater may be used.

Methods for transposon tagging can be used to reduce or eliminate thelevel of at least one seed protein in grain. The methods of transposontagging comprise insertion of a transposon within an endogenous plantseed gene to reduce or eliminate expression of the seed protein.

Methods for transposon tagging of specific genes in plants are wellknown in the art (see, for example, Maes, et al., (1999) Trends PlantSci. 4:90-96; Dharmapuri and Sonti, (1999) FEMS Microbiol. Lett.179:53-59; Meissner, et al., (2000) Plant J. 22:265-274; Phogat, et al.,(2000) J. Biosci. 25:57-63; Walbot, (2000) Curr. Opin. Plant Biol.2:103-107; Gai, et al., (2000) Nuc. Acids Res. 28:94-96; Fitzmaurice, etal., (1999) Genetics 153:1919-1928). In addition, the TUSC process forselecting Mu insertions in selected genes has been described (Bensen, etal., (1995) Plant Cell 7:75-84; Mena, et al., (1996) Science274:1537-1540; U.S. Pat. No. 5,962,764, which is herein incorporated byreference).

Other methods for inhibiting or eliminating the expression of endogenousgenes are also known in the art and can be similarly applied to theinstant invention. These methods include other forms of mutagenesis,such as ethyl methanesulfonate-induced mutagenesis, deletionmutagenesis, and fast neutron deletion mutagenesis used in a reversegenetics sense (with PCR) to identify plant lines in which theendogenous gene has been deleted (for examples of these methods seeOhshima, et al., (1998) Virology 243:472-481; Okubara, et al., (1994)Genetics 137:867-874; Quesada, et al., (2000) Genetics 154:421-436. Inaddition, a fast and automatable method for screening for chemicallyinduced mutations, TILLING, (Targeting Induced Local Lesions InGenomes), using a denaturing HPLC or selective endonuclease digestion ofselected PCR products is also applicable to the instant invention (see,McCallum, et al., (2000) Nat. Biotechnol. 18:455-457).

Mutation breeding is another of many methods that could be used tointroduce new traits into an elite line. Mutations that occurspontaneously or are artificially induced can be useful sources ofvariability for a plant breeder. The goal of induced mutagenesis is toincrease the rate of mutation for a desired characteristic. Mutationrates can be increased b many different means including: temperature;long-term seed storage; tissue culture conditions; radiation such asX-rays, Gamma rays (e.g., Cobalt 60 or Cesium 137), neutrons, (productof nuclear fission by Uranium 235 in an atomic reactor, Beta radiation(emitted from radioisotopes such as P32, or C14) or ultravioletradiation (preferably from 2500 to 2900 nm) or chemical mutagens such asbase analogues (5-bromo-uracil), related compounds (8-ethoxy caffeine),antibiotics (streptonigrin), alkylating agents (sulfur mustards,nitrogen mustards, epoxides, ethylenamines, sulfates, sulfonates,sulfones, lactones), azide, hydroxylamine, nitrous acid or acridines.Once a desired trait is observed through mutagenesis, the trait may thenbe incorporated into existing germplasm by traditional breedingtechniques, such as backcrossing. Details of mutation breeding can befound in “Principals of Cultivar Development” Fehr, 1993 (MacmillanPublishing Company), the disclosures of which are incorporated herein byreference. In addition, mutations created in other lines may be used toproduce a backcross conversion of elite lines that comprise suchmutations.

Other methods for inhibiting or eliminating the expression of genesinclude the transgenic application of transcription factors (Pabo, etal., (2001) Annu Rev Biochem 70:313-40 and Reynolds, et al., (2003) ProcNatl Acad Sci USA 100:1615-20) and homologous recombination methods forgene targeting (see, U.S. Pat. No. 6,187,994).

Similarly, it is possible to eliminate the expression of a single geneby replacing its coding sequence with the coding sequence of a secondgene using homologous recombination technologies (see, Bolon, (2004)Basic Clin. Pharmacol. Toxicol. 95(4)(12):154-61; Matsuda and Alba,(2004) Methods Mol. Bio. 259:379-90; Forlino, et. al., (1999) J. Biol.Chem. 274(53):37923-30). For example, by using the knock-out/knock-intechnology, the coding sequence of a UXS polypeptide can be replaced bythe coding sequence of a closely related polypeptide resulting insuppression of UXS protein expression and in over-expression of theclosely related protein.

In some embodiments, the polynucleotide expressed by the expressioncassette of the invention is catalytic RNA or has ribozyme activityspecific for the messenger RNA of a protein of interest. Thus, thepolynucleotide causes the degradation of the endogenous messenger RNA,resulting in reduced expression of one or more proteins. This method isdescribed, for example, in U.S. Pat. No. 4,987,071, herein incorporatedby reference.

In some embodiments of the invention, the polynucleotide comprises aninhibitory sequence that encodes an antibody that binds to at least oneisoform of a seed protein and reduces the level of the seed protein. Inanother embodiment, the binding of the antibody results in increasedturnover of the antibody-antigen complex by cellular quality controlmechanisms. The expression of antibodies in plant cells and theinhibition of molecular pathways by expression and binding of antibodiesto proteins in plant cells are well known in the art. See, for example,Conrad and Sonnewald, (2003) Nature Biotech. 21:35-36, incorporatedherein by reference

Plant transformants containing a desired genetic modification as aresult of any of the above described methods resulting in increased,decreased or eliminated expression of the seed protein of the inventioncan be selected by various methods known in the art. These methodsinclude, but are not limited to, methods such as SDS-PAGE analysis,immunoblotting using antibodies which bind to the seed protein ofinterest, single nucleotide polymorphism (SNP) analysis or assaying forthe products of a reporter or marker gene, and the like.

Plant Cell Transformation

Transformation protocols as well as protocols for introducing nucleotidesequences into plants can vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include, but are not limitedto: microinjection (Crossway, et al., (1986) Biotechniques 4:320-334);electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA83:5602-5606); Agrobacterium-mediated transformation (Townsend, et al.,U.S. Pat. No. 5,563,055; Zhao, et al., U.S. Pat. No. 5,981,840; Cai, etal., U.S. patent application Ser. No. 09/056,418); direct gene transfer(Paszkowski, et al., (1984) EMBO J. 3:2717-2722) and ballistic particleacceleration (see, for example, Sanford, et al., U.S. Pat. No.4,945,050; Tomes, et al., U.S. Pat. No. 5,879,918; Tomes, et al., U.S.Pat. No. 5,886,244; Bidney, et al., U.S. Pat. No. 5,932,782; Tomes, etal., (1995) “Direct DNA Transfer into Intact Plant Cells viaMicroprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin)and McCabe, et al., (1988) Biotechnology 6:923-926). Also see,Weissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al.,(1987) Particulate Science and Technology 5:27-37 (onion); Christou, etal., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen, (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor.Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563(maize); Tomes, U.S. Pat. No. 5,240,855; Buising, et al., U.S. Pat. Nos.5,322,783 and 5,324,646; Tomes, et al., (1995) “Direct DNA Transfer intoIntact Plant Cells via Microprojectile Bombardment,” in Plant Cell,Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg(Springer-Verlag, Berlin) (maize); Klein, et al., (1988) Plant Physiol.91:440-444 (maize); Fromm, et al., (1990) Biotechnology 8:833-839(maize); Hooykaas-Van Slogteren, et al., (1984) Nature (London)311:763-764; Bowen, et al., U.S. Pat. No. 5,736,369 (cereals); Bytebier,et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); DeWet, et al., (1985) in The Experimental Manipulation of Ovule Tissues,ed. Chapman, et al., (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler, etal., (1990) Plant Cell Reports 9:415-418 and Kaeppler, et al., (1992)Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation);D'Halluin, et al., (1992) Plant Cell 4:1495-1505 (electroporation); Li,et al., (1993) Plant Cell Reports 12:250-255 and Christou and Ford,(1995) Annals of Botany 75:407-413 (rice); Osjoda, et al., (1996) NatureBiotechnology 14:745-750 (maize via Agrobacterium tumefaciens), all ofwhich are herein incorporated by reference.

The methods of the invention can involve introducing a nucleotideconstruct into a plant. By “introducing” is intended presenting to theplant the nucleotide construct in such a manner that the construct gainsaccess to the interior of a cell of the plant. The claimed methods donot depend on a particular method for introducing a nucleotide constructto a plant, only that the nucleotide construct gains access to theinterior of at least one cell of the plant. Methods for introducingnucleotide constructs into plants are known in the art including, butnot limited to, stable transformation methods, transient transformationmethods and virus-mediated methods.

By “stable transformation” is intended that the nucleotide constructintroduced into a plant integrates into the genome of the plant and iscapable of being inherited by progeny thereof. By “transienttransformation” is intended that a nucleotide construct introduced intoa plant does not integrate into the genome of the plant.

The nucleotide constructs of the invention may be introduced into plantsby contacting plants with a virus or viral nucleic acids. Generally,such methods involve incorporating a nucleotide construct of theinvention within a viral DNA or RNA molecule. It is recognized that theprotein of interest of the invention may be initially synthesized aspart of a viral polyprotein, which later may be processed by proteolysisin vivo or in vitro to produce the desired recombinant protein. Further,it is recognized that promoters of the invention also encompasspromoters utilized for transcription by viral RNA polymerases. Methodsfor introducing nucleotide constructs into plants and expressing aprotein encoded therein, involving viral DNA or RNA molecules, are knownin the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190,5,866,785, 5,589,367 and 5,316,931, herein incorporated by reference.

Other methods of transformation include Agrobacteriumrhizogenes-mediated transformation (see, e.g., Lichtenstein and FullerIn: Genetic Engineering, vol. 6, Rigby, Ed., London, Academic Press,1987; and Lichtenstein and Draper, In: DNA Cloning, Vol. II, Glover,Ed., Oxford, IRI Press, 1985), Application PCT/US87/02512 (WO 88/02405published Apr. 7, 1988) discloses the use of A. rhizogenes strain A4 andits Ri plasmid along with A. tumefaciens vectors pARC8 or pARC16

The introduction of DNA constructs using PEG precipitation is disclosedin Paszkowski, et al., (1984) EMBO J. 3:2717-2722. Electroporationtechniques are disclosed in Fromm, et al., (1985) Proc. Natl. Acad. Sci.(USA) 82:5824. Ballistic transformation techniques are disclosed inKlein, et al., (1987) Nature 327:70-73. Alternative transformationmethods include liposome-mediated DNA uptake (see, e.g., Freeman, etal., (1984) Plant Cell Physiol. 25:353) and the vortexing method (see,e.g., Kindle, (1990) Proc. Natl. Acad. Sci. (USA) 87:1228.

DNA can also be introduced into plants by direct DNA transfer intopollen as disclosed by Zhou, et al., (1983) Methods in Enzymology101:433; Hess, (1987) Intern Rev. Cytol. 107:367; Luo, et al., (1988)Plant Mol. Biol. Reporter 6:165. Expression of polypeptide coding genescan be obtained by injection of the DNA into reproductive organs of aplant as disclosed by Pena, et al., (1987) Nature 325:274. DNA can alsobe injected directly into the cells of immature embryos and therehydration of desiccated embryos as disclosed by Neuhaus, et al.,(1987) Theor. Appl. Genet. 75:30 and Benbrook, et al., in ProceedingsBio Expo 1986, Butterworth, Stoneham, Mass., pp. 27-54 (1986). A varietyof plant viruses that can be employed as vectors are known in the artand include cauliflower mosaic virus (CaMV), geminivirus, brome mosaicvirus and tobacco mosaic virus. The claimed methods and compositions arenot limiting as to the method of transformation and any such planttransformation method known in the art can be utilized.

Transgenic Plant Regeneration

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype and/or phenotype. See, forexample, McCormick, et al., (1986) Plant Cell Reports 5:81-84,incorporated herein by reference. These plants can then be grown andeither pollinated with the same transformed strain or different strainsand the resulting hybrid having expression of the desired phenotypiccharacteristic identified. Two or more generations can be grown toensure that expression of the desired phenotypic characteristic isstably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.

Plants cells transformed with a plant expression cassette can beregenerated, e.g., from single cells, callus tissue or leaf discsaccording to standard plant tissue culture techniques. Various cells,tissues, and organs can be successfully cultured to regenerate an entireplant. Plant regeneration from cultured protoplasts is disclosed inEvans, et al., Protoplasts Isolation and Culture, Handbook of Plant CellCulture, Macmillan Publishing Company, New York, pp. 124-176 (1983) andBinding, Regeneration of Plants, Plant Protoplasts, CRC Press, BocaRaton, pp. 21-73 (1985).

The regeneration of plants containing a foreign gene introduced byAgrobacterium from leaf explants can be achieved as disclosed by Horsch,et al., (1985) Science 227:1229-1231. In this procedure, transformantsare grown in the presence of a selection agent and in a medium thatinduces the regeneration of shoots in the plant species beingtransformed. These transformed shoots are then transferred to anappropriate root-inducing medium containing a selective agent and anantibiotic to prevent bacterial growth.

Regeneration can also be obtained from plant callus, explants, organs orparts thereof. The regeneration of plants from either single plantprotoplasts or various explants is well known in the art. See, forexample, Methods for Plant Molecular Biology, Weissbach and Weissbach,eds., Academic Press, Inc., San Diego, Calif. (1988). This regenerationand growth process includes the steps of selection of transformant cellsand shoots, rooting the transformant shoots and growth of the plantletsin soil. For maize cell culture and regeneration see generally, TheMaize Handbook, Freeling and Walbot, Eds., Springer, N.Y. (1994); Cornand Corn Improvement, 3.sup.rd edition, Sprague and Dudley Eds.,American Society of Agronomy, Madison, Wis. (1988).

The desired genetically altered trait can be bred into other plant linespossessing desirable agronomic characteristics using conventionalbreeding methods and/or top-cross technology. The top-cross method istaught in U.S. Pat. No. 5,704,160 herein incorporated in its entirety byreference.

Methods for cross pollinating plants are well known to those skilled inthe art and are generally accomplished by allowing the pollen of oneplant, the pollen donor, to pollinate a flower of a second plant, thepollen recipient, and then allowing the fertilized eggs in thepollinated flower to mature into seeds. Progeny containing the entirecomplement of heterologous coding sequences of the two parental plantscan be selected from all of the progeny by standard methods available inthe art for selecting transformed plants. If necessary, the selectedprogeny can be used as either the pollen donor or pollen recipient in asubsequent cross pollination.

Parts obtained from the regenerated plant, such as flowers, seeds,leaves, branches, fruit, and the like are included in the claimedmethods and compositions, provided that these parts comprise cellscomprising the disclosed nucleic acids of the invention. Progeny andvariants and mutants of the regenerated plants are also included withinthe scope of the claimed subject matter, provided that these partscomprise the introduced nucleic acid sequences.

Transgenic plants expressing a selectable marker can be screened fortransmission of one or more target nucleic acids by, for example,standard immunoblot and DNA detection techniques. Transgenic lines arealso typically evaluated on levels of expression of the heterologousnucleic acid. Expression at the RNA level can be determined initially toidentify and quantitate expression-positive plants. Standard techniquesfor RNA analysis can be employed and include PCR amplification assaysusing oligonucleotide primers designed to amplify only the heterologousRNA templates and solution hybridization assays using heterologousnucleic acid-specific probes. The RNA-positive plants can be analyzedfor polypeptide expression by Western immunoblot analysis usingspecifically or selectively reactive antibodies. In addition, in situhybridization and immunocytochemistry according to standard protocolscan be done using heterologous nucleic acid specific polynucleotideprobes and antibodies, respectively, to localize sites of expressionwithin transgenic tissue. Generally, a number of transgenic lines areusually screened for the incorporated nucleic acid to identify andselect plants with the most appropriate expression profiles.

The claimed methods and/or compositions further provides for modulating(i.e., increasing or decreasing) the concentration or ratio ofpolypeptides, such as UXS polypeptides, in a plant or part thereof.Modulation can be effected by increasing or decreasing the concentrationand/or the ratio of the polypeptides in a plant.

EXAMPLES

The following examples are included to illustrate various embodiments ofthe invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered to function well in the practice of the claimedmethods. However, those of skill in the art should, in light of thepresent disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Example 1 UDP-Xylose Synthase Isoforms

Eight UXS isoforms were identified and named UXS1 through 8. Amino acidsequences of the maize isoforms are provided in the sequence listing asSEQ ID NOS: 2, 4, 6, 8, 10, 12, 14 and 16. Nucleic acid sequencesencoding the UXS isoforms are provided in the sequence listing as SEQ IDNOS: 1, 3, 5, 7, 9, 11, 13 and 15. The eight isoforms can be generallyclassified into three distinct types. Type A includes ZmUXS1 and 2,orthologous to AtUXS3, 5, 6, which are likely soluble and cytosoliclocalized. Type B includes ZmUXS3 and 4, orthologous to AtUXS1. Type Cincludes ZmUXS5, 6, 7, and 8, orthologous to AtUXS2 and 4. Both type Band C contain a trans-membrane domain and are likely Golgi localized.

Expression levels of different UXS isoforms were determined in differentmaize tissues using standard techniques. Briefly, the eight UXS DNAsequences were used to blast an MPSS tag library and unique tags wereidentified for each isoform (Massively Parallel Signature Sequencing)technology from LYNX™ (see, Brenner, et al, (2000) Nature Biotechnology18:630-634). The unique and 3′ most tags for each isoform were used toblast the MPSS profiling database. The average abundance of the tags wascalculated from LYNX libraries for each tissue, with the expressionlevels of the UXS isoforms taken from the relative abundance of thecorresponding tags.

UXS expression in maize endosperm showed a different developmentalpattern from other seed tissues, with a decrease in total UXS expressionobserved between 6 DAP (days after pollination) and 40 DAP. The isoformprofile shifted from primarily zmUXS1, zmUXS2 and zmUXS6 at DAP6 to analmost equal, but low, mixture of isoforms 1, 2, 3, 4, 6 and 8 at DAP40.

In contrast, maize pericarp showed an increase in total UXS expressionfrom DAP 5 to DAP 27. However the dominant isoforms in pericarp did notvary much with time after pollination, being primarily zmUXS1 andzmUXS2, with a lesser amount of zmUXS6 at DAP 5. Although the totalexpression level increases at DAP 27, the same three isoformspredominated. A comparison of various maize tissues showed that zmUXS1,zmUXS2 and zmUXS6 tended to be the predominant isoforms expressed inmaize, although the relative levels varied from tissue to tissue.ZmUXS3, zmUXS4 and zmUXS8 appear to be minor alleles in general inmaize.

Example 2 Agrobacterium-Mediated Transformation of Maize

For Agrobacterium-mediated transformation of maize, nucleotide sequencesof interest such as UXS sequences were operably linked to a promoter asdisclosed below employing the transformation method of Zhao, et al.(U.S. Pat. No. 5,981,840 and PCT Patent Publication WO98/32326, thecontents of which are hereby incorporated by reference). Briefly,immature embryos were isolated from maize and the embryos contacted witha suspension of Agrobacterium, where the bacteria were capable oftransferring the nucleotide sequence of interest to at least one cell ofat least one of the immature embryos (infection). In this step theimmature embryos were immersed in an Agrobacterium suspension for theinitiation of inoculation. The embryos were co-cultured for a time withthe Agrobacterium (co-cultivation). The immature embryos were culturedon solid medium following infection. Following this co-cultivationperiod an optional “resting” stage was performed. In this resting stage,the embryos were incubated in the presence of at least one antibioticknown to inhibit the growth of Agrobacterium without the addition of aselective agent for plant transformants (resting). The immature embryoswere cultured on solid medium with antibiotic, but without a selectingagent, for elimination of Agrobacterium and for a resting phase for theinfected cells. Next, inoculated embryos were cultured on mediumcontaining a selective agent and growing transformed callus wasrecovered (selection). The immature embryos were cultured on solidmedium with a selective agent resulting in the selective growth oftransformed cells. The callus was then regenerated into plants and calligrown on selective medium were cultured on solid medium to regeneratethe plants.

Example 3 Analyzing Seed for Hemicellulose Content

For a given ear, dissected endosperms were pooled into wild type ortransgenic based on Western blot results. The pooled endosperm tissuewas ground and weighed out into samples in duplicate 2 ml screw topmicrofuge tubes for hemicellulose preparations.

Removal of Soluble Sugars:

Small stir bars were placed in each tube and 1 ml 80% ethanol added andvortexed to mix. Tubes were heated in heat block set at 100° C. for 30seconds and again vortexed to mix.

Tubes were then centrifuged at 14,000 rpm for 10 minutes and thesupernatant discarded using vacuum. 1 ml of acetone was added and tubesvortexed to mix.

After another centrifugation at 14,000 rpm for 10 minutes, supernatantwas again discarded using vacuum and pellets left in hood to dry (about4 hours).

De-Starching:

0.3 ml of α-amylase solution (300 U/assay amylase in MOP buffer) wasadded to the tubes and mixed by vortexing. Tubes were then placed inracks and immersed in hot water. Heating proceeded at 90-95° C. for 15minutes with constant stirring on magnetic stir plate. Samples werecooled slightly and briefly spun down to remove moisture bycentrifugation (1 minute). At this point, 0.2 ml of 285 mM Na-acetatecontaining AMG (20 U/assay) mix was added and tubes incubated at 55° C.for 4 hours or overnight followed by a brief spin down by centrifugation(1 minute) to remove moisture.

Washing:

1.25 ml of absolute ethanol was added to each tube (bringing finalethanol concentration to 70%). Samples were vortexed and cooled on iceor in −20 freezer for 10-15 minutes.

Three rounds of centrifugation at 14,000 rpm for 10 minutes after whichthe supernatant was discarded using vacuum and 1 ml 80% ethanol addedand vortexed between rounds. After the last round of centrifugation, thesupernatant was vacuum removed and discarded and 1 ml acetone added,vortexed, and centrifuged at 14,000 rpm for 10 minutes. This supernatantwas also removed using vacuum and discarded. The pellets were thenallowed to air-dry in fume hood.

Hydrolyzing and Quantifying Hemicellulose:

1 ml of 1 M H₂SO₄ was added to air-dried tubes and vortexed to mix.Samples were then heated in 100° C. heat block for 45 minutes andvortexed every few minutes to mix. Samples were then cooled on ice.

Samples were centrifuged at 14,000 rpm for 10 minutes and 0.6 ml ofsupernatant removed into new tubes for HPLC.

Sugar residues from hemicellulose were quantified by HPLC DionexCarboPac PA-1, 4×250 mm analytical column, PN 35391 Dionex PAD, withpulsed amperometric detector.

Individual hemicellulosic sugar residue was expressed as percent ofendosperm weight. Arabinoxylan is the sum of arabinose and xylose % ofcontrol e.g.: arabinoxylan (transgenic)/arabinoxylan (wild type) X 100.

Example 4 Down-Regulation of UDP-Xylose Synthases in Grain to ReduceHemicellulose

Based on expression profiles, members of Type A (ZmUXS1 and 2) and TypeC (ZmUXS5, 6, 7 and 8) showed relatively high levels of expression inall three tissues of grain (pericarp, endosperm and embryo), whilemembers of Type B (ZmUXS3 and 4) had apparently low endogenousexpression in grain. Because of the high degree of nucleotide sequencehomology between UXS1 and 2 and between UXS5, 6, 7 and 8, RNAiconstructs of UXS2 and UXS6 under the control of promoters specific forpericarp, endosperm and embryo are expected to result in silencingexpression of both groups of UXS isoforms. In turn, it is expected thata reduction of hemicellulose content in maize kernel will occur,improving grain digestibility and increasing efficiency of ethanoloutput per unit grain in dry milling.

As demonstrated in the case of UDP-GDH, (U.S. Pat. No. 6,399,859) apractical approach to reduce hemicellulose content can comprise blockingnucleotide sugar biosynthesis. UDP-xylose synthase (UXS) can also beused for this purpose because the reaction UXS catalyzes is downstreamof UDP-GDH and additionally bypasses the myo-inositol pathway.Therefore, blocking UXS should provide potent and specific inhibition ofhemicellulose production by inhibiting UDP-xylose synthesis.

The following constructs were prepared for co-suppression of UXSexpression in maize. Promoters on each end of the expression cassettedirect the insert to be expressed in different tissues at differenttimes if desired. The cassettes contain no termination signals. Thecassette included a selectable marker gene such as PAT (Wohlleben, etal., (1988) Gene 70:25-37 or BAR for resistance toBasta/phosphinothricin.

Methods of construction of such expression cassettes is well known tothose of skill in the art in light of the present disclosure. See, forexample, e.g., Sambrook, et al., (1989) Molecular Cloning: A LaboratoryManual (Cold Spring Harbor, Laboratory Press, Plainview, N.Y.; Gelvin,et al., (1990) Plant Molecular Biology Manual, each incorporated hereinin its entirety by reference.

Table of Abbreviations Abbreviation Element Name Reference GZPRO 27 kDgamma zein promoter Reina, et al., (1990) Nucleic Acid Res. 18(21): 6426RFP DsRed2 Clontech Laboratories, Mountain View, CA OLE Oleosin promoterIRNT inverted repeat, no terminator N/A LTP2 PRO barley LTP2 promoterKalla, et al., (1994) Plant Journal 4: 849-860 LEG1A PRO legumin1promoter US Patent Application Publication Number 2006/0130184 ZM-40 PROZea mays-40 promoter U.S. Pat. No. 6,403,862 EAP1 PRO EAP1 promoter U.S.Pat. No. 7,081,566 LTP1 PRO LTP1 promoter U.S. patent application Ser.No. 11/408,223

Construct PHP19991 (see, SEQ ID NO: 17 for insert sequence) was preparedto down-regulate the UXS 6 sequence. However, because of the high levelof homology with UXS5 and UXS7, it was expected PHP19991 should inhibitexpression of UXS 5, 6 and 7, but not UXS8. Maize was transformed withPHP19991 by Agrobacterium-mediated transformation as described herein.The genotypes of T1 kernels from the same event were screened with PCR,and the endosperms pooled into WT or transgenic. Hemicellulose analysiswas performed with pooled endosperms as described herein.

PHP19991: GZ PRO:UXS6 IRNT:OLE PRO

Construct and sequence homology:

PHP19991/UXS6F vs UXS1=47.1%

PHP19991/UXS6F vs UXS2=48.2%

PHP19991/UXS6F vs UXS3=55.8%

PHP19991/UXS6F vs UXS4=57.3%

PHP19991/UXS6F vs UXS5=90.5%

PHP19991/UXS6F vs UXS6=100%

PHP19991/UXS6F vs UXS7=97.9%

PHP19991/UXS6F vs UXS8=65.9%

The effects of co-suppression using the PHP19991 cassette were to reducethe arabinoxylan contents in transgenic endosperm by up to about 20% inthe T1 generation. The results demonstrate that UXS 5, 6 and/or 7contribute part of the UDP-xylose substrate for hemicellulose synthesis.

Construct PHP21179 (see, SEQ ID NO: 18 for insert) was prepared todown-regulate the UXS2 sequence. Because of the high level of homology,PHP21179 was expected to inhibit expression of UXS 1 and 2 (Type A), thesoluble isoforms of UXS in maize. Maize was transformed with PHP21179 byAgrobacterium-mediated transformation as described herein. The genotypesof T1 kernels from the same event were screened with PCR, and theendosperms pooled into WT or transgenic. Hemicellulose analysis wasperformed with pooled endosperms.

PHP21179: LEG1A PRO:Uxs2 IRNT ZM-40 PRO:Uxs2 (IRNT)

Construct and sequence homology:

PHP21179/UXS2F vs UXS1=94.2%

PHP21179/UXS2F vs UXS2=100%

PHP21179/UXS2F vs UXS3=65.7%

PHP21179/UXS2F vs UXS4=65.5%

PHP21179/UXS2F vs UXS5=66.2%

PHP21179/UXS2F vs UXS6=65.7%

PHP21179/UXS2F vs UXS7=66.1%

PHP21179/UXS2F vs UXS8=65.5%

The arabinoxylan contents in transgenic endosperm transformed withPHP21179 also showed up to about a 20% reduction in the T1 generation.The results show that UXS1 and 2 also play a role in producingUDP-xylose for hemicellulose synthesis.

Construct PHP21180 (ZM-40 PRO:Uxs2 IRNT::EAP1B PRO:Uxs2 IRNT) wasprepared and transformed into maize by Agrobacterium-mediatedtransformation, as discussed above. T0 events of PHP21180 were selfed.Hence only 25% of the seeds would be expected to be wild-type in asegregating ear and 25% would be homozygous transgenic.

Twenty seeds from 12 events of PHP21180 were dissected to separateendosperm, embryo and pericarp. Twenty mg of ground endosperm was PCRgenotyped to identify wild type and transgenic seeds using MOPATprimers. Nine out of the 12 events that segregated as single copy werefurther used for cell wall analysis. Embryo from seeds that segregatedas wild type and transgenic seeds were pooled for each event. Foursamples of 15-25 mg were used for cell wall sugar analysis. Reduction ofarabinoxylan contents in transgenic embryos of up to 50% of controllevels were observed.

Construct PHP21807 (see, SEQ ID NO: 19 for insert sequence) was preparedto inhibit both Type A and Type C isoforms of UXS. Based on sequencecomparisons, this construct was expected to down-regulate UXS 1, 2, 5,6, 7 and possibly UXS 8. PHP21807 was transformed into maize byAgrobacterium-mediated transformation. The genotypes of T1 kernels fromthe same event were screened with PCR and the endosperms pooled into WTor transgenic. Hemicellulose analysis was performed with pooledendosperms as described herein.

PHP21807:LEG1APRO:[Uxs2::Uxs6] IRNT ZM-40 PRO:[Uxs2::Uxs6] IRNT

Construct and sequence homology:

PHP21807/UXS2F vs UXS1=94.2%

PHP21807/UXS2F vs UXS2=100%

PHP21807/UXS2F vs UXS3=65.7%

PHP21807/UXS2F vs UXS4=65.5%

PHP21807/UXS2F vs UXS5=66.2%

PHP21807/UXS2F vs UXS6=65.7%

PHP21807/UXS2F vs UXS7=66.1%

PHP21807/UXS2F vs UXS8=65.5%

PHP21807/UXS6F vs UXS1=65.3%

PHP21807/UXS6F vs UXS2=66.2%

PHP21807/UXS6F vs UXS3=71.9%

PHP21807/UXS6F vs UXS4=72.0%

PHP21807/UXS6F vs UXS5=95.4%

PHP21807/UXS6F vs UXS6=100%

PHP21807/UXS6F vs UXS7=99.2%

PHP21807/UXS6F vs UXS8=85.6%

Transformation with PHP21807 resulted in a slightly greater reduction inarabinoxylan content in transgenic endosperm in the T1 generation,compared to either PHP19991 or PHP21179 alone, with up to about a 25%decrease observed.

Although the UXS sequences, enzyme activities and cellular localizationshave been studied in plant species other than maize, their involvementin hemicellulose biosynthesis has not previously been demonstrated.These results provide the first demonstration that inhibiting expressionof UXS causes a reduction in plant hemicellulose accumulation. Theresults also demonstrate that both the soluble and membrane bound UXSplay a role in producing UDP-xylose for hemicellulose synthesis.

Example 5 Additional Co-suppression Studies of UDP-Xylose Synthases

T2 generation seeds were produced by back-crossing transgenic T1 plants.The presence of UXS nucleic acid sequences were determined by PCRanalysis, using construct specific primers. Arabinoxylan content wasdetermined in pooled samples as described in Example 3. Maize T2endosperm transformed with PHP19991, PHP21179 and PHP21807 all showedreduced hemicellulose content, with a greater reduction in arabinoxylancontent observed in the T2 endosperm compared to T1 endosperm.

PHP23388: LTP1 PRO:RFP::GZ PRO:UXS2-UXS6 IRNT:OLE PRO

A stacked construct containing sequences targeted against Type A andType C isoforms was prepared and transformed into maize. T1 endospermcontaining the construct were analyzed for hemicellulose, cellulose andhemicellulosic galactose content. Inhibition of two types of UXSisoforms resulted in a greater decrease in hemicellulose content thaninhibition of Type A alone or Type C alone.

This construct contained a different promoter than PHP21807: LTP1, apericarp preferred promoter. The insert sequence in PHP23388 was thesame as in PHP21807. PHP23388 was transformed into maize byAgrobacterium-mediated transformation as described herein. Hemicellulosecontent of T1 endosperm was determined as described above.

Construct PHP23388 resulted in a greater inhibition of hemicellulosecontent than PHP21807 as well as an unexpected reduction in celluloseand hemicellulosic galactose. Hemicellulose was reduced up to 50% inboth endosperm and embryo with the majority of events having reductionsof 20% or more. Cellulose was reduced up to 25% in both embryo andendosperm with the majority of events having at least a 10% reduction inboth tissues. Embryo galactose was reduced by at least 20% in themajority of events and endosperm galactose reduced by at least 10′)/0 inmost events.

LTP2 PRO:RFP::GZ PRO: UXS2-UXS6-UXS3-UXS8 IRNT: OLE PRO

LTP2 PRO:RFP::GZ PRO: UXS3-UXS8 IRNT: OLE PRO

These constructs are transformed by Agrobacterium transformation intomaize as described above. T1 seed are analyzed and compared to wild-typeto determine the function of Type B UXS.

LTP1 PRO: UXS2-UXS6-UXS3-UXS8 IRNT

LTP1 PRO: ADH1 INTRON: UXS2-UXS6 IRNT

These constructs are transformed by Agrobacterium transformation intomaize as described above. T1 seed are analyzed and compared to wild-typeand other transformants to determine the efficacy and effects of UXS onpericarp to achieve greater reduction of fiber in seed.

Example 6 Agrobacterium-Mediated Transformation of Sorghum

For Agrobacterium-mediated transformation of sorghum the method of Cai,et al., is employed (U.S. patent application Ser. No. 09/056,418, thecontents of which are hereby incorporated by reference). This method canbe employed with any of the nucleotide sequences described above.

Example 7 Transformation of Maize Embryos by Particle Bombardment

Immature maize embryos from greenhouse donor plants are bombarded with aplasmid containing any of the nucleotide sequences disclosed above,operably linked to a selected promoter, plus a plasmid containing theselectable marker gene PAT (Wohlleben, et al., (1988) Gene 70:25-37)that confers resistance to the herbicide Bialaphos. Transformation isperformed as follows.

Preparation of Target Tissue

The ears are surface sterilized in 30% Clorox bleach plus 0.5% Microdetergent for 20 minutes and rinsed two times with sterile water. Theimmature embryos are excised and placed embryo axis side down (scutellumside up), 25 embryos per plate, on 560Y medium for 4 hours and thenaligned within the 2.5-cm target zone in preparation for bombardment.

Preparation of DNA

A plasmid cassette comprising the nucleotide sequence of interestoperably linked to a promoter is made. This plasmid DNA plus plasmid DNAcontaining a PAT selectable marker is precipitated onto 1.1 μm (averagediameter) tungsten pellets using a CaCl₂ precipitation procedure asfollows:

-   -   100 μl prepared tungsten particles in water    -   10 μl (1 μg) DNA in Tris EDTA buffer (1 μg total)    -   100 μl 2.5 M CaCl₂    -   10 μl 0.1 M spermidine

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 ml 100% ethanol and centrifugedfor 30 seconds. Again the liquid is removed, and 105 μl 100% ethanol isadded to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μlspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

Particle Gun Treatment

The sample plates are bombarded at level #4 in particle gun #HE34-1 or#HE34-2.

All samples receive a single shot at 650 PSI, with a total of tenaliquots taken from each tube of prepared particles/DNA.

Subsequent Treatment

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/literBialaphos and subcultured every 2 weeks. After approximately 10 weeks ofselection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for the desired phenotypic trait.

Bombardment and Culture Media

Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMAC-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/lthiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline(brought to volume with D-I H₂0 following adjustment to pH 5.8 withKOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H₂0) and8.5 mg/l silver nitrate (added after sterilizing the medium and coolingto room temperature). Selection medium (560R) comprises 4.0 g/l N6 basalsalts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511),0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought tovolume with D-I H₂0 following adjustment to pH 5.8 with KOH); 3.0 g/lGelrite (added after bringing to volume with D-I H₂0); and 0.85 mg/lsilver nitrate and 3.0 mg/l bialaphos (both added after sterilizing themedium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL and 0.40 g/l glycinebrought to volume with polished D-I H₂0) (Murashige and Skoog, (1962)Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/lsucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume withpolished D-I H₂0 after adjusting to pH 5.6); 3.0 g/l Gelrite (addedafter bringing to volume with D-I H₂0) and 1.0 mg/l indoleacetic acidand 3.0 mg/l bialaphos (added after sterilizing the medium and coolingto 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinicacid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/lglycine brought to volume with polished D-I H₂0), 0.1 g/1 myo-inositoland 40.0 g/l sucrose (brought to volume with polished D-I H₂0 afteradjusting pH to 5.6) and 6 g/l bacto-agar (added after bringing tovolume with polished D-I H₂0), sterilized and cooled to 60° C.

Example 8 Transformation of Rice Embryogenic Callus by Bombardment

Embryogenic callus cultures derived from the scutellum of germinatingseeds serve as the source material for transformation experiments. Thismaterial is generated by germinating sterile rice seeds on a callusinitiation media (MS salts, Nitsch and Nitsch vitamins, 1.0 mg/l 2,4-Dand 10 μM AgNO₃) in the dark at 27-28° C. Embryogenic callusproliferating from the scutellum of the embryos is then transferred toCM media (N6 salts, Nitsch and Nitsch vitamins, 1 mg/1 2,4-D, Chu, etal., (1985) Sci. Sinica 18:659-668). Callus cultures are maintained onCM by routine sub-culture at two week intervals and used fortransformation within 10 weeks of initiation.

Callus is prepared for transformation by subculturing 0.5-1.0 mm piecesapproximately 1 mm apart, arranged in a circular area of about 4 cm indiameter, in the center of a circle of Whatman #541 paper placed on CMmedia. The plates with callus are incubated in the dark at 27-28 C for3-5 days. Prior to bombardment, the filters with callus are transferredto CM supplemented with 0.25 M mannitol and 0.25 M sorbitol for 3 hr. inthe dark. The petri dish lids are then left ajar for 20-45 minutes in asterile hood to allow moisture on tissue to dissipate.

Circular plasmid DNA from two different plasmids, one containing theselectable marker for rice transformation and one containing a nucleicacid of interest, are co-precipitated onto the surface of goldparticles. To accomplish this, a total of 10 μg of DNA at a 2:1 ratio oftrait:selectable marker DNAs is added to a 50 μl aliquot of goldparticles resuspended at a concentration of 60 mg/ml. Calcium chloride(50 μl of a 2.5 M solution) and spermidine (20 μl of a 0.1 M solution)are then added to the gold-DNA suspension as the tube is vortexing for 3min. The gold particles are centrifuged in a microfuge for 1 sec and thesupernatant removed. The gold particles are then washed twice with 1 mlof absolute ethanol and then resuspended in 50 μl of absolute ethanoland sonicated (bath sonicator) for one second to disperse the goldparticles. The gold suspension is incubated at −70 C for five minutesand sonicated (bath sonicator) if needed to disperse the particles. Sixμl of the DNA-coated gold particles are then loaded onto mylarmacrocarrier disks and the ethanol is allowed to evaporate.

At the end of the drying period, a petri dish containing the tissue isplaced in the chamber of the PDS-1000/He. The air in the chamber is thenevacuated to a vacuum of 28-29 inches Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1080-1100 psi. Thetissue is placed approximately 8 cm from the stopping screen and thecallus is bombarded two times. Five to seven plates of tissue arebombarded in this way with the DNA-coated gold particles. Followingbombardment, the callus tissue is transferred to CM media withoutsupplemental sorbitol or mannitol.

Within 3-5 days after bombardment the callus tissue is transferred to SMmedia (CM medium containing 50 mg/l hygromycin). To accomplish this,callus tissue is transferred from plates to sterile 50 ml conical tubesand weighed. Molten top-agar at 40° C. is added using 2.5 ml of topagar/100 mg of callus. Callus clumps are broken into fragments of lessthan 2 mm diameter by repeated dispensing through a 10 ml pipet. Threeml aliquots of the callus suspension are plated onto fresh SM media andthe plates incubated in the dark for 4 weeks at 27-28° C. After 4 weeks,transgenic callus events are identified, transferred to fresh SM platesand grown for an additional 2 weeks in the dark at 27-28° C.

Growing callus is transferred to RM1 media (MS salts, Nitsch and Nitschvitamins, 2% sucrose, 3% sorbitol, 0.4% gelrite+50 ppm hyg B) for 2weeks in the dark at 25° C. After 2 weeks the callus is transferred toRM2 media (MS salts, Nitsch and Nitsch vitamins, 3% sucrose, 0.4%gelrite+50 ppm hyg B) and placed under cool white light (˜40 μEm⁻²s⁻¹)with a 12 hr photoperiod at 25° C. and 30-40% humidity. After 2-4 weeksin the light, callus generally begins to organize, and form shoots.Shoots are removed from surrounding callus/media and gently transferredto RM3 media (½×MS salts, Nitsch and Nitsch vitamins, 1% sucrose+50 ppmhygromycin B) in phytatrays (Sigma Chemical Co., St. Louis, Mo.) andincubation is continued using the same conditions as described in theprevious step.

Plants are transferred from RM3 to 4″ pots containing Metro mix 350after 2-3 weeks, when sufficient root and shoot growth has occurred.Plants are grown using a 12 hr/12 hr light/dark cycle using ˜30/18° C.day/night temperature regimen.

Example 9 Transformation of Dicots

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage polypeptide phaseolin from the bean Phaseolus vulgaris(Doyle, et al., (1986) J. Biol. Chem. 261:9228-9238) can be used forexpression of nucleic acids in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), SmaI, KpnI and XbaI. Theentire cassette is flanked by Hind III sites.

The cDNA fragment of this gene can be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression cassette. Amplification is then performed and the isolatedfragment is inserted into a pUC18 cassette carrying the seed expressioncassette.

Soybean embryos can then be transformed with the expression cassettecomprising nucleic acid sequences as disclosed herein. To induce somaticembryos, cotyledons, 3-5 mm in length dissected from surface sterilized,immature seeds of the soybean cultivar A2872, can be cultured in thelight or dark at 26° C. on an appropriate agar medium for 6-10 weeks.Somatic embryos which produce secondary embryos are then excised andplaced into a suitable liquid medium. After repeated selection forclusters of somatic embryos which multiplied as early, globular stagedembryos, the suspensions are maintained as described below.

Soybean embryogenic suspension cultures can maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures can then be transformed by themethod of particle gun bombardment (Klein, et al., (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont BiolisticPDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a nucleic acid composed of the 35S promoter fromCauliflower Mosaic Virus (Odell, et al., (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz, et al., (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, a nucleic acid insert of interest and the phaseolin 3′ regioncan be isolated as a restriction fragment. This fragment can then beinserted into a unique restriction site of the cassette carrying themarker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media can be exchangedwith fresh media and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue can be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line can be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 10 Expression of a Nucleic acid in Microbial Cells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression cassette pBT430. This cassette is a derivative ofpET-3a (Rosenberg, et al., (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 isconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites is inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression cassette. Then, the Nde I site at the positionof translation initiation is converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, is converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA can be appropriately digested to release anucleic acid fragment encoding the polypeptide. This fragment can thenbe purified on a 1% NuSieve GTG low melting agarose gel (FMC). Bufferand agarose contain 10 μg/ml ethidium bromide for visualization of theDNA fragment. The fragment can then be purified from the agarose gel bydigestion with GELase (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters can be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The cassette pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared cassette pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21(DE3) (Studier, et al., (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°C. Cells are then harvested by centrifugation and re-suspended in 50 μLof 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the polypeptideconcentration of the supernatant determined. One microgram ofpolypeptide from the soluble fraction of the culture can be separated bySDS-polyacrylamide gel electrophoresis. Gels can be observed forpolypeptide bands migrating at the expected molecular weight.

Example 11 Preparation of Transgenic Sunflowers

Biolistics/Bombardment

Mature sunflower seeds are dehulled and surface sterilized for 30 min ina 20 percent Chlorox® bleach solution with the addition of two drops ofTween 20 per 50 ml of solution. Seeds are rinsed twice with distilledwater.

Seeds are imbibed in distilled water for 60 min following the surfacesterilization procedure. The cotyledons of each seed are broken off toproduce a clean fracture at the plane of the embryonic axis. Followingexcision of the root tip, the explants are bisected longitudinallybetween the primordial leaves. The two halves are placed cut surface upon GBA medium consisting of Murashige and Skoog mineral elements,Shepard's vitamin additions, 40 mg/l adenine sulfate, 30 g/l sucrose,0.5 mg/l 6-benzyl-aminopurine, 0.25 mg/l indole-3-acetic acid, 0.1 mg/lgibberellic acid pH 5.6, and 8 g/l Phytagar.

Thirty to forty explants at a time are placed in a circle at the centerof a 60×20 mm plate for microprojectile bombardment. Approximately 4.7mg of 1.8 um tungsten microprojectiles are re-suspended in 25 ml ofsterile TE buffer (10 mM Tris-Cl, 1 mM EDTA pH 8) and 1.5 m aliquots areused per bombardment. Each plate is bombarded twice through a 150 umNytex screen placed 2 cm above the samples in a PDS 1000® particleacceleration device.

Agro Transformation

Plasmid is introduced into Agrobacterium tumefaciens via freeze thawingas described by Holsters, et al., (1978) Mol. Gen. Genet. 163:181-7.Bacteria used for transformation are grown overnight (28° C. and 100 RPMcontinuous agitation) in liquid YEP medium (10 g/l yeast extract, 10 g/lBactopeptone and 5 g/l NaCl, pH 7.0) in the presence of kanamycin. Thesuspension is used when it reached an OD600 of 0.5 in an incubationmedium comprised of 12.5 mM 2-(N-morpholino) ethanesulfonic acid, MES, 1g/l NH4Cl and 0.3 g/l MgSO4 at pH 5.7.

Freshly bombarded explants are placed in an Agrobacterium suspension,mixed and left undisturbed for 30 min. The explants are then transferredto GBA medium with co-cultivated cut surfaces down at 26° C. for 18-hdays. After 3 days of co-cultivation, the explants are transferred to374B media (GBA medium lacking growth regulators and having a reducedsucrose level of 1 percent) supplemented with 250 mg/l cefotaxime and25, 50, 100 or 200 mg/l kanamycin sulfate. The explants are cultured for2-5 weeks on the supplemented medium and then transferred to fresh 374Bmedium lacking kanamycin for 1-2 weeks of continued development.Explants with differentiating, antibiotic resistant areas of growth thathad not produced shoots suitable for excision are transferred to GBAmedium containing 250 mg/l cefotaxime for a second 3-day phytohormonetreatment. Leaf samples from green, kanamycin-resistant shoots areassayed for the presence of selectable marker activity. Those shootsthat fail to exhibit activity are discarded.

Marker positive shoots are grafted to Pioneer® hybrid in vitro-grownsunflower seedling rootstock. Surface sterilized seeds are germinated in48-0 medium (half strength Murashige and Skoog salts, 0.5 percentsucrose, 0.3 percent gelrite pH 5.6) and grown under the conditionsdescribed for explant culture. The upper portion of each seedling isremoved, a 1-cm vertical slice is made in each hypocotyl, and eachtransformed shoot is inserted into a cut. The entire area of eachprepared graft is wrapped with parafilm to secure each shoot. Graftedplants are transferred to soil following 1 week of in vitro culture.Grafts in soil are maintained under high humidity conditions, followedby a slow acclimatization to a greenhouse environment.

Transformed sectors of T0 plants (parental generation) maturing in thegreenhouse are identified by marker analysis of leaf extracts whiletransgenic seeds harvested from marker-positive T0 plants are identifiedby analysis of small portions of dry seed cotyledon. Leaf and seedassays of confirmed transgenics also are performed for segregationanalysis of selfed progeny populations. Transgenic seeds from a numberof events are cultivated under field conditions. The transgenics aregrown in an isolation cage designed to minimize pollen dissemination byforaging insects.

DNA is isolated from immature leaves of greenhouse grown sunflowerplants by a urea buffer extraction protocol. For each sample, leaftissue (2 g) is frozen in liquid nitrogen, ground with a mortar andpestle and mixed into 6 ml of urea extraction buffer (50 mM TRIS-HCl pH8.0, 7M urea, 0.31 M NaCl, 1 percent sarcosine). An equal volume ofphenol:chloroform (1:1) is added and the mixture is shaken at roomtemperature for 15 minutes. After centrifugation (12,000×g, 15 min) theclarified supernatant is removed. DNA is precipitated by the addition of1 ml of 4.4 M ammonium acetate (pH 5.2) and 7 ml isopropanol. The DNA iscollected with a Pasteur pipette hook, allowed to dry for 15 minutes,and then resuspended in 0.5 M TE buffer as described in Ausubel, et al.,CURRENT PROTOCOLS 1N MOLECULAR BIOLOGY, (1989). DNA (about 10 mg) isdigested twice with 5-10 units of XbaI/mg DNA at 370 for 3 hours.Samples are subjected to electrophoresis on a one percent agarose geland transferred onto nylon membranes for hybridization analysis asdescribed by Southern, (1975) J. Mol. Biol. 98: 503-17.

Appropriate enzyme restriction digestion linearizes the binary plasmid.T-DNA junctions with plant genomic DNA at the right border are detectedusing restriction enzyme digestion followed by hybridization to aradiolabeled probe generated by random prime labeling of the markerfragment as described by Feinberg and Vogelstein, (1984) Anal. Biochem.137: 266-7.

REFERENCES

-   Bar-Peled M, Griffith C L, Doering T L (2001) Functional cloning and    characterization of a UDPglucuronic acid decarboxylase: the    pathogenic fungus Cryptococcus neoformans elucidates UDP-xylose    synthesis. Proc Natl Acad Sci USA 98:12003-12008-   Gu X, Bar-Peled M (2004) The biosynthesis of UDP-galacturonic acid    in plants: functional cloning and characterization of Arabidopsis    UDP-D-galacturonic acid 4-epimerase. Plant Physiol 136:4256-4264-   Harper A D, Bar-Peled M (2002) Biosynthesis of UDP-Xylose. Cloning    and characterization of a novel Arabidopsis gene family, UXS,    encoding soluble and putative membrane-bound UDP-Glucuronic acid    decarboxylase isoforms. Plant Physiol 130:2188-2198-   Hayashi T, Koyama T, Matsuda K (1988) Formation of UDP-xylose and    xyloglucan in soybean Golgi membranes. Plant Physiol 87:341-345-   Kobayashi M, Nakagawa H, Suda I, Miyagawa I, Matoh T (2002)    Purification and cDNA cloning of UDP-D-glucuronate carboxy-lyase    (UDP-D-xylose synthase) from pea seedlings. Plant Cell Physiol    43:1259-1265-   Moriarity J L, Hurt K J, Resnick A C, Storm P B, Laroy W, Schnaar R    L, Snyder H (2002) UDP-glucoronate decarboxylase, a key enzyme in    proteoglycan synthesis. J Biol Chem 277:16968-16975-   Pagny S, Bouissonnie F, Sarkar M, Follet-Gueye M L, Driouich A,    Schachter H, Faye L, Gomord V (2003) Structural requirements for    Arabidopsis beta1,2-xylosyltransferase activity and targeting to the    Golgi. Plant J 33:189-203-   Pattathil S, Harper A D, Bar-Peled M (2005) Biosynthesis of    UDP-xylose: characterization of membrane-bound AtUxs2. Planta. Jan    18-   Reiter W D, Vanzin G F (2001) Molecular genetics of nucleotide sugar    interconversion pathways in plants. Plant Mol Biol 47:95-113-   Seifert G J (2004) Nucleotide sugar interconversion and cell wall    biosynthesis: how to bring the inside to outside. Curr Opin Plant    Biol 7:277-284-   Suzuki K, Suzuki Y, Kitamura S (2003) Cloning and expression of a    UDP-glucuronic acid decarboxylase gene in rice. J Exp Bot    54:1997-1999-   Wheatley E R, Davies D R, Bolwell G P (2002) Characterisation and    immunolocation of an 87 kDa polypeptide associated with    UDP-glucuronic acid decarboxylase activity from differentiating    tobacco cells (Nicotiana tabacum L.). Phytochemistry 61:771-780

What is claimed is:
 1. An expression cassette comprising an isolatedpolynucleotide comprising a member selected from the group consistingof: (a) a polynucleotide having at least 95% sequence identity to SEQ IDNO: 18 wherein the % sequence identity is determined over the entirelength of the sequence by the GAP algorithm using default parameters;wherein the isolated polynucleotide modulates the level of arabinoxylan;and (b) a polynucleotide comprising SEQ ID NO: 18, wherein the isolatedpolynucleotide is operably linked to a heterologous promoter.
 2. Theexpression cassette of claim 1 further comprising any combination ofadditional polynucleotide sequences of interest.
 3. A plant cellcontaining the expression cassette of claim
 1. 4. A transformed plantcomprising the expression cassette of claim
 1. 5. The transformed plantof claim 4, wherein the plant is corn, barley, soybean, sorghum, wheat,rice, alfalfa, safflower, sunflower, canola, cotton or millet.
 6. Atransformed seed from the transformed plant of claim
 4. 7. A method formodulating arabinoxylan levels in a plant cell, comprising: (a)transforming the host cell with the expression cassette of claim 1; and(b) growing the transformed host cell to modulate arabinoxylan levels inthe plant cell.
 8. The method of claim 7, further comprisingtransforming the plant cell with any combination of additionalpolynucleotide sequences of interest.
 9. The method of claim 7, furthercomprising producing a transformed plant from the plant cell.